|Detection of Human Phospho-GSK‑3 beta (S9) by Western Blot. Western blot shows lysates of HeLa human cervical epithelial carcinoma cell line untreated (-) or treated (+) with 200 nM PMA for 20 minutes. PVDF Membrane was probed with 1 µg/mL of Human Phospho-GSK‑3 beta (S9) Monoclonal Antibody (Catalog # MAB25062) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # HAF007). A specific band was detected for Phospho-GSK‑3 beta (S9) at approximately 46 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.|
|Phospho-GSK‑3 beta (S9) in HeLa Human Cell Line. GSK‑3 beta phosphorylated at S9 was detected in immersion fixed HeLa human cervical epithelial carcinoma cells, unstimulated (lower panel) or stimulated (upper panel) with PMA, using Mouse Anti-Human Phospho-GSK‑3 beta (S9) Monoclonal Antibody (Catalog # MAB25062) at 10 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Mouse IgG Secondary Antibody (red; Catalog # NL007) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm. View our protocol for Fluorescent ICC Staining of Cells on Coverslips.|
Intracellular Staining by Flow Cytometry
|Detection of Phospho-GSK-3 beta (S9) in HeLa Human Cell Line by Flow Cytometry. HeLa human cervical epithelial carcinoma cell line untreated (open histogram) or treated with 120 ng/mL PMA for 15 minutes (filled histogram) was stained with Human Phospho-GSK‑3 beta (S9) Monoclonal Antibody (Catalog # MAB25062) or isotype control antibody (Catalog # MAB002, not shown), followed by Fluorescein-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # F0103B). To facilitate intracellular staining, cells were fixed with paraformaldehyde and permeabilized with saponin.|
Glycogen Synthase Kinase-3 (GSK-3) is a serine/threonine kinase initially identified asan inhibitor of glycogen synthase. Two isoforms (GSK-3 alpha and GSK-3 beta ) share 85% amino acid identity. GSK-3 beta, inhibited by phosphorylation at S9 by Akt, is involved in energy metabolism, body pattern formation, and neuronal cell development.